Fig. 2

Design of the recombination-based mutation barcoding (ReMB) library and its application to functional screening. a The design of the ReMB library and its screening schema are illustrated. We constructed a series of donor clones containing the gene-coding sequences of wild-type or various mutations according to the mutation profiles of the TCGA, COSMIC, and FUSCC cohorts. A retroviral pDEST-HA-Flag vector was used to construct the barcode plasmids. Random 30-bp annealed oligonucleotide pairs were inserted into this retroviral backbone. We then constructed a set of vectors containing various barcodes with one barcode in each vector. The recombination reaction was performed for a retroviral mutation plasmid that contained a unique barcode sequence. All of the clones were mixed into a pool to package a gene mutations retrovirus library that included mutants, wild-type and negative controls. The gene mutation library was delivered into MCF-10A or HEMC cells by retroviral infection at an MOI of 0.3. Cells stably expressing the mutated genes were obtained by puromycin selection for 5 days. Functional screens were conducted for 2 weeks to examine proliferation, doxorubicin, and BKM120 responses, followed by PCR amplification of the barcode sequences that were integrated into the chromosomes. The purified PCR products were subjected to barcode deconvolution analysis using NGS. b Growth curves were obtained for transduced MCF-10A cells that were exposed to concentrations of doxorubicin (1.5 nM) or BKM120 (1.2 µM) for 14 days. Five wells were measured per condition. The error bars indicate mean ± s.d. derived from three independent experiments. c Schematic showing the primer design. d Electrophoresis revealed a DNA fragment containing 30-bp barcodes after PCR; Sanger sequencing was used to validate the mixed barcode sequences